Novel microfluidic device for factor VIII quantification by chemiluminescence in hemophilia A patients

Abstract

Point-of-care (PoC) testing can revolutionize diagnostics, enabling early detection and disease monitoring for chronic disorders. However, current PoC systems often lack the adaptability to detect multiple analytes. We present a capillary-based microfluidic detection platform with integrated optical sensors, that allows for an array of 16 simultaneous measurements using a chemiluminescent readout. The device consists of a disposable part containing microfluidics and a reusable part with integrated optical sensors. To demonstrate proof-of-principle, the current study specifically focuses on quantifying clotting Factor VIII (FVIII), a critical parameter in Hemophilia A management. Therefore, a chemiluminescent-based FVIII assay (FVIII_lum) was designed to quantify the activity levels in citrated plasma samples. All reagents necessary for the assay are contained in the disposable part of the device. To detect FIXa/FVIIIa tenase activity, a caged chemiluminescent substrate specific for activated Factor X (FXa) was synthesized for ultrasensitive real-time measurement of FVIII-mediated FXa generation. Experiments with the FVIII_lum, performed with the microfluidic cartridge with dried-in reagents, showed high level of precision. In addition, the FVIII_lum was capable to distinguish FVIII deficient plasma from plasma with only 0.017 IU/mL activity, demonstrating its applicability for Hemophilia A management. Finally, proof-of-principle was obtained by pharmacokinetic monitoring of FVIII activity in patient samples, demonstrating good agreement with the reference method. This study successfully presents a new miniaturized chemiluminescent platform, incorporating microfluidics and photon sensors, designed to detect multiple parameters in parallel per cartridge.